Radioimmunoassay (also referred to as RIA) is a very sensitive method for the determination of antigens or antibodies, both qualitative and quantitative. Quantities down to 10.sup.-.sup.12 gram can be detected. The method is based upon the competition between radiolabelled and unlabelled antigen for a fixed and limited amount of antibody as described by R. Yalow et al in J. Clin. Invest., 39, 1157 (1960). The amount of unlabelled antigen influences the distribution of the labelled antigen in antibody-bound and antibody-free labelled antigen. That is, the more unlabelled antigen which is present, the less labelled antigen gets the opportunity to combine with antibody.
In the accompanying drawings, FIGS. 1 to 3 are standard curves obtained by measuring standard concentrations of designated antigens in accordance with the present invention. FIGS. 4 and 5 illustrate a test tube assay means in accord with the present invention.
The term "kit" is employed herein to mean a collection of all or some of the chemicals, including the assay tubes, and instructions necessary to do a radioimmunoassay.
The term "antibody" or "antibodies" is employed to mean a group of serum proteins, also referred to as gamma globulins or immunoglobulins, that will specifically react with an antigen or hapten. Most of these antibodies belong to the IgG class, while the other classes are termined IgA, IgM, IgD, and IgE. It is also used herein to include certain binding proteins which recognize certain antigens, for example such protein for cyclic AMP and for cortisol.
The term "antigen" is employed herein to means a substance that will react with an antibody. Antigens are often characterized as capable of inducing the formation of an antibody and of reacting with that antibody. However, in the case of "haptens" it is necessary to be coupled to a carrier, such as, for example, inert adsorbing particles, synthetic peptides, or natural protein molecules, in order to induce antibody formation. Materials commonly employed as carriers include for example, the albumins (human, bovine, or rabbit), synthetic peptides (for example, polylysine), inert adsorbing particles (for example, dextran-coated charcoal) and polymers (for example, polyvinylpyrrolidone). However, haptens will in the absence of a carrier still react with antibodies and can be employed in the antigen-antibody reaction assays of the present invention either with or without carriers.
A necessary requirement for obtaining conclusive results from the radioimmunoassay method is the separation between the antibody-bound and antibody-free antigen. The development of various solid phase methods provides for rapid and efficient separation. In the solid phase type of assay, the antibody or antigen is bonded or fixed to an adsorbent, which facilitates separation or precipitation of bound or free fractions. See, for example K. J. Catt, U.S. 3,646,346 and K. Catt et al, Science, 158, 1570 (1967).
The solid phase antibody techniques make use of antibodies covalently bonded or fixed to insoluble polymers, convalently cross-linked, or physically adsorbed to a material, such as, for example, plastics, glass, or freshly deposited metals. The insoluble carriers or immunoadsorbents include bentonite particles, bromoethyl cellulose, the crosslinked dextrans (Sephadex), and beaded agarose (Sepharose).
While large amounts of antibodies are required in the solid phase antibody systems, they do have certain advantages. Binding of the antigen is rapid, virtually irreversible, and the two phases may be separated by simple decantation or low-speed centrifugation. In many instances antibody solutions can be reused for adsorption.
Polymerized antibodies, covalently cross-linked with ethyl chloroformate or glutaraldehyde, provide a practical and accurate method for assays. However, the method requires high speed centrifugation for precipitation of the antigen-antibody complex.
The solid-phase antibody method by bonding-adsorption includes the use of poly(tetrafluoroethylene-g-aminostyrene) ("Protapol") powder, poly (tetrafluoroethylene-g-isothicyanatostyrene) powder ("Protapol DI/1"), or polystyrene plastic tubes. The preparation of the antibody-coated discs or tubes may be affected by the pH, molarity, temperature, and protein concentration of the incubating solution. However the technique permits many determinations to be performed rapidly, and it is highly sensitive and reproducible.
One modification of the solid phase method, described by T. Goodfriend et al, Immunochemistry, 6, 484 (1969), uses an acrylamide gel that seems to entrap the antibody, but allows diffusion of low-molecular-weight antigens.
Additional information concerning radioimmunoassy is set forth in D. S. Shelly et al Clin. Chem., 19, 146 (1973).
While the radioimmunoassay is a valuable technique in clinical practice, its use has been relatively limited. One possible reason for this is that it is difficult to set up, especially for a non-routine assay. In addition, commercial kits currently available are often unreliable, have poor stability and short shelf lives, are intricate and involved to the point of requiring skilled technicians for their operation.
It is an object of the present invention to provide a convenient means for the immunoassay of a component of the antibody antigen reaction. It is a further object to provide an improved kit for the radioimmunoassay method, free from the drawbacks of the prior art methods mentioned hereinabove.
It has surprisingly been found that the objects of the present invention can preferably be accomplished by introducing all of the reagents necessary for a radioimmunoassay into a single tube in a manner such that the antibody is bound to at least a part of the surface and the antigen is dispersed in solid form over at least a part of the surface but not bound thereto nor to the antibody.
The unitized solid phase radioimmunoassay kit offers several advantages over other radio-immunoassay kits. These include (1) elimination of the necessity for the technician to have to dilute to pipet radioactive material in combining the components in the assay procedure, which will result in increased safety for the technician, (2) the elimination of the necessity for the technician to have to dilute or pipet any material except the patient's sample which will result in a decreased chance of error and time spent doing the assay and (3) increased stability and storage life. Therefore, the unitized solid phase radioimmunoassay kit of the present invention offers potential savings in laboratory technician time, kit cost to a laboratory, and test cost to a patient.